Introduction: Hydrogen-deuterium exchange (HDX) mass spectrometry (MS) is ideal for monitoring the protein folding and unfolding. The exchange of a deuterium in solution for an amide hydrogen in a protein can be very different depending on the degree of folding and protection of backbone amide positions. Molecular chaperones that assist with protein folding in vivo are necessary for folding of many substrate (client) proteins. HDX MS provides valuable insight into what chaperones are doing in protein folding and how they are doing it. Areas covered: Application of HDX MS to the protein folding problem was desirable from the outset of the technique, but technical issues prohibited many studies. In the last 20 years, conformational changes of chaperones themselves (e.g., GroEL/GroES, Hsp70, and Hsp90) have been studied. Studies of interactions between chaperones, co-chaperones, and substrate proteins have revealed binding interfaces, allosteric conformational changes, and remodeling of components during various chaperone cycles. Experiments elucidating how chaperones contribute to and enhance the folding pathway of substrate proteins have been demonstrated. Expert opinion: Technical issues that once prevented the analysis of chaperones have largely been resolved, permitting exciting comprehensive HDX MS studies of folding pathways during chaperone-assisted protein folding.
Keywords: GroEL/GroES; HDX MS; Hsp70; Hsp90; TRiC/CCT; chaperonin; conformational changes; protein-protein interaction.